Material for promoting skin basement formation

ABSTRACT

A method for promoting skin basement membrane formation comprises administering matrix metalloproteinase inhibitor or matrix metalloproteinase inhibitor and matrix protein production promoting agent. The matrix metalloproteinase inhibitor is N-hydroxy-2(R)-[[(4-methoxyphenyl)sulfonyl](3-picolyl)amino]-3-methylbutanamide hydrochloride.

This application claims the priority of Japanese Patent Application No.2000-87574, filed on Mar. 27, 2000, which is incorporated herein byreference. This application is a continuation of U.S. patent applicationSer. No. 10/648,485, filed on Aug. 27, 2003, which is a continuation ofU.S. patent application Ser. No. 09/979,712, filed on Nov. 26, 2001 andlater abandoned (International Patent Application Number PCT/JP200102507filed Mar. 27, 2001).

FIELD OF THE INVENTION

The present invention relates to a skin basement membrane stabilizingagent. In addition, the present invention relates to artificial skinformation promoting agent and a production method for artificial skin.

BACKGROUND ART

In the field of cosmetics and dermatology, a wide variety of measureshave been proposed and attempted to alleviate or treat injuries causedby the effects of the external environment and aging including theexposure of the skin to sunlight. For example, examples of skin changesaccompanying aging primarily include the formation of wrinkles, andhardening or decreased resiliency.

Primary interest is being directed at decreased function of collagenfibers and elastic fibers composed of collagen, elastin andglucosaminoglycans in the skin dermis as the cause of such changes. Inthe past, the use of hydroxycarboxylic acids (e.g., Japanese Patent No.2533339), and the use of lysophospholipids (e.g., Japanese UnexaminedPatent Publication No. 8-67621) or that described in the Journal of theJapan Society of Fats and Oils, Vol. 46, No. 9 (1997), pp. 13-19 havebeen proposed as means for preventing or repairing such changes.

In the former publication, keratin and wrinkles have been suggested asbeing able to be eradicated by preventing decreases in collagen fibers.On the other hand, in the latter publication, lysophospholipids aresuggested to demonstrate whitening effects by accelerating theproduction ability of glycosaminoglycans (and more specifically,hyaluronic acid) in human fibroblasts.

The most powerful effect of the above external environment on skin agingis produced by ultraviolet rays present in sunlight, and theseultraviolet rays have clearly been established to be a factor thatpromotes aging. Ultraviolet rays are known to induce skin changesreferred to as photoaging that is characterized by deep wrinkles(Scharffetter-Kochanek, Advances in Pharmacology, 1997, 38, 639-655).Ultraviolet rays have a diverse range of effects on the skin, includingdamage to genetic DNA, induced production of active oxygen, and morerecently, induced production of matrix-metalloproteinases (Fisher, etal., Nature, 1996, 379, 335-339).

Due to the multifunctional nature of ultraviolet rays, the mechanism bywhich this photoaging induced by ultraviolet rays occurs has not beenadequately elucidated. Deep wrinkles corresponding to human photoagedskin have been clearly shown to form on the skin on the backs ofhairless mice following continuous irradiation of ultraviolet rays at anenergy level not enough to cause erythema. Substances having an effecton wrinkles have also been evaluated using this mouse model (Moloney, etal., Photochem. Photobiol. 1992, 56, 495-504). However, the wrinkleformation mechanism has still not been adequately elucidated and itselucidation is awaited.

On the other hand, Koivukangas, et al. reported in 1994 that levels ofgelatinase, an enzyme that decomposes the basement membrane, becomeselevated in skin irradiated with ultraviolet rays (Acta Derm. Venereol.1994, 74, 279-282). In addition, it has also been reported that thebasement membrane exhibits structural changes at sites of the skinexposed to sunlight, and that reduplication is observed particularlyfrequently (Lavker, J. Invest. Dermal. 1979, 73, 59-66). This suggeststhe possibility that ultraviolet rays contained in sunlight have aneffect on basement membrane structure by increasing the amount ofbasement membrane decomposing enzymes produced in the skin.

However, there is no specific means known for promoting the formation ofskin basement membrane structure.

Artificial skin is important as an alternative to natural skin that hasbeen damaged by some cause, or as an experimental material for testingthe action and pharmacological effects of pharmaceuticals and cosmeticson the skin. In either of these applications, artificial skin is desiredthat has a structure that imitates the structure of natural skin asclosely as possible.

Natural skin is roughly composed of the epidermis, dermis and basementmembrane present between them. One example of a known production methodof artificial skin that imitates natural skin involves the formation ofan epidermal layer by culturing normal human epidermal keratinocytes oncontracted type I collagen gel having a structure that resembles thedermis and contains human fibroblasts. In this method, however, therewas the problem of inadequate formation of a basement membrane betweenthe collagen gel imitating the dermis and the epidermal layer imitatingthe epidermis.

DISCLOSURE OF THE INVENTION

Thus, the object of the present invention is to provide a novel meansfor stabilizing skin basement membrane, and to provide a novel means forachieving adequate formation of basement membrane in the production ofartificial skin.

As a result of various studies to solve the above problems, theinventors of the present invention found that the formation of a skinbasement membrane structure is promoted by administering matrixmetalloproteinase inhibitor, or both matrix metalloproteinase inhibitorand matrix protein production promoting agent, thereby leading tocompletion of the present invention.

Thus, the present invention provides a skin basement membrane formationpromoting agent that contains matrix metalloproteinase inhibitor.

In addition, the present invention provides a skin basement membraneformation promoting agent containing matrix metalloproteinase inhibitorand matrix protein production promoting agent.

As a result of various studies to solve the above problems, theinventors of the present invention found that, in culturing to produceartificial skin, formation of a basement membrane can be promoted byadding matrix metalloproteinase inhibitor, or both matrixmetalloproteinase inhibitor and matrix protein production promotingagent to the medium, thereby leading to completion of the presentinvention.

Thus, the present invention provides an artificial skin formationpromoting agent containing matrix metalloproteinase inhibitor.

In addition, the present invention provides an artificial skin formationpromoting agent containing matrix metalloproteinase inhibitor and matrixprotein production promoting agent.

In addition, the present invention provides an artificial skinproduction method comprising the addition of matrix metalloproteinaseinhibitor to an artificial skin formation medium.

Moreover, the present invention provides an artificial skin productionmethod comprising the addition of matrix metalloproteinase inhibitor andmatrix protein production promoting agent to an artificial skinformation medium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph representing a biological form of thecross-section of artificial skin comparing the case of addition ofmatrix metalloproteinase CGS27023A (10 μM) or MMP inhibitor (300 μM)with a control to which neither is added in culturing for the formationof artificial skin.

FIG. 2 is a photograph representing a biological form of thecross-section of artificial skin that shows the results of performinghematoxylin and eosin staining (H & E) and immunostaining (detection oflaminin (LN5)) on formed artificial skin in the case of adding matrixmetalloproteinase inhibitor CGS27023A (shown as CGS) only, the case ofadding CGS and transforming growth factor α, a matrix protein productionpromoting agent (CGS+TGFa), the case of adding CGS and transforminggrowth factor β1, a matrix protein production promoting agent(CGS+TGFb1), and the case of adding both TGFa and TGFb1 to CGS(CGS+TGFa+TGFb1) to the medium during culturing for formation ofartificial skin.

FIG. 3 is a photograph used in place of a drawing representing abiological form of a fragment of artificial skin showing the results ofperforming immunostaining (detection of type IV collagen (IVC) anddetection of type VII collagen (VIIC)) in the same experiment as FIG. 2.

FIG. 4 is a photograph representing a biological form of cross-sectionsof artificial skin formed in the case of adding matrix metalloproteinaseinhibitor CGS27023A and soy bean lecithin, a matrix protein productionpromoting agent (CGS+soy bean lecithin), and the case of not adding theabove substances (control) to the medium during culturing for theformation of artificial skin.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

Matrix Metalloproteinase Inhibitor

There are no particular restrictions on the matrix metalloproteinaseinhibitor used in the present invention, and any substance may be usedprovided it has such inhibitory activity. Examples of matrixmetalloproteinase include gelatinase, collagenase, stromelysin andmatrilysin. Thus, a substance (inhibiting) such as gelatinase,collagenase, stromelysin or matrilysin can be selected for the matrixmetalloproteinase inhibitor.

Specific examples of matrix metalloproteinase inhibitors includesubstance CGS27023AN-hydroxy-2(R)-[[(4-methoxyphenyl)sulfonyl](3-picolyl)amino]-3-methylbutanamidehydrochloride (J. Med. Chem. 1997, Vol. 40, p. 2525-2532), andMMP-inhibitor (p-NH₂-Bz-Gly-Pro-D-Leu-Ala-NHOH) (FN-437) (BBRC, 1994,Vol. 199, p. 1442-1446).

Moreover, specific examples of metalloproteinase inhibitor of thepresent invention that can be used include various plant extracts andpurified products obtained therefrom. Examples of such plants includeThymus serpyllum L., Valeriana faureie Briquet or other similar plants(Valerianaceae), Diospyros kaki Thunberg (Ebenaceae), Astragalus sinicusLinne (Leguminosae) Crataegus cuneata Siebold et Zuccarini (Rosaceae),Paeonia suffruticosa Andrews (Poeonia montan Sims) (Paconiaceae), Theasinensis Linne var. assamica Pierre (Thcaccae), Eucalyptus globulusLabillardiere or its similar plants (Myrtaceae), Potentilla tormentillaSchrk (Rosaceae), Tilia corda: a Mill., Tilia platyphyllus Scop., Tiliaeuropaea Linne (Tiliaceae), Betula alba Linne (Betulaceze),Origanummajorana L., Uncaria gambir Roxburgh (Rubiaceae), Juglans regiaLinne var. sinensis De Candolie or its similar plants (Juglandaceae),Sophora flavescens Aiton (Leguminosae), Sanguisorba officinalis Linne(Rosaceae), Hypericum perforatum Linne or Hypericum erectum Thunberg(Guttiferae), Thea sinensis Linne (Theaceae), Curcuma longa L(Zingiberaceae), purified extracts of Curcuma longa L includingSymplocos racemosa and Cyperus rotundus, Cyperus scariosus, Gaultheriafragrantissima, Acacia fornensia, Terminalia chebula, Ficus bengalensis,Cassia fistula Linn, Lyonia ovalifolia, Calophyllum inophyllum and Ficusreligiosa.

Extracts of these plants are obtained from the root, leaves, stem orflower in the case of herbaceous plants, or from the root, buds, bark,fruits, leaves or flowers in the case of woody plants.

Extracts from these plants are obtained by drying the plant material asnecessary and cutting or crushing as necessary, followed by extractinginto an aqueous extracting agent or organic solvent. Examples of aqueousextracting agents that can be used include cold water, warm water andhot water at the boiling point or lower temperature, while examples oforganic solvents that can be used include methanol, ethanol,1,3-butanediol and ether at normal temperature or heated.

Examples of matrix proteins in the present invention include laminin,type IV collagen, type VII collagen, parlecan and nidogen, which areconstituent components of the basement membrane.

Matrix Protein Production Promoting Agent

Examples of the matrix protein production promoting agent used in thepresent invention include, in the case of promoting the production ofthese proteins, soy bean lysolecithin, transforming growth factor α(TGFα), transforming growth factor β1 (TGFb1), transforming growthfactor β2 (TGFb2), transforming growth factor β3 (TGFb3) and epitheliumgrowth factor (EGF).

In the case the artificial skin formation promoting agent or skinbasement membrane stabilizing agent of the present invention containsmatrix metalloproteinase inhibitor and matrix protein productionpromoting agent, the ratio of matrix metalloproteinase inhibitor andmatrix protein production promoting agent, although varying according totheir types and activity, is roughly 1:10⁶ to 10⁶:1.

Artificial Skin

Any arbitrary medium that is conventionally used for the production ofartificial skin can be used for the basal medium used to produceartificial skin in the present invention, and examples of such mediainclude Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetalcalf serum, DMEM-Ham's F12 (3:1) medium containing 10% fetal calf serum,5 μg/ml of transferrin, 5 μg/ml of insulin, 2 nM tri-iodotyrosine, 0.1nM cholera toxin and 0.4 μg/ml of hydrocortisone, and keratinocytegrowth medium (KGM) and DMEM containing 10% fetal calf serum mixed at aratio of 1:1. Although varying according to the particular type, theamount of matrix metalloproteinase inhibitor added to these basal mediais about 1 nmol/L to 10⁻² mole/L. In addition, the amount of matrixprotein production promoting agent added to the basal medium is about 1ng/L to 1 g/L.

In the production of artificial skin of the present invention,contracted type I collagen gel containing human fibroblasts is firstallowed to stand undisturbed on a metal mesh. The contracted type Icollagen containing human fibroblasts can be prepared in, for example,the manner described below. After preparing a collagen solution in whichfibroblasts are suspended over ice, the collagen is gelled in a Petridish. Next, the gel is peeled from the walls of the Petri dish and thecollagen gel is contracted in a CO₂ incubator.

Next, epidermal cells, such as normal human epidermal keratinocytes, arecultured on the above collagen gel to form epidermal. Formation of anepidermal layer by culturing epidermal cells can be carried out in thefollowing manner. Contracted collagen gel is placed on a metal mesh, anda glass ring is placed on this gel. A suspension of epidermalkeratinocytes originating in human foreskin is placed in the glass ringwhile preventing liquid leaking. The keratinocytes are adhered in a CO₂incubator, followed by removal of the ring. The above medium is filledto the boundary of the epidermal layer and cornified layer is formed bycontinuing culturing while exposing the epidermal layer to the air.

According to this method, artificial skin is obtained that approximatesnatural skin in which an adequate basement membrane is formed between adermal layer and epidermal layer composed of contracted type I collagengel containing fibroblasts.

Skin Basement Membrane Formation Promoting Agent

According to the result of a study on basement membrane structuralchanges of facial skin for which cosmetics are primarily used thatfocused on a report of structural changes characterized by reduplicationof the basement membrane accompanying aging as one of the changes thataccompany aging (Lavker, J., Invest. Dermal. 1979, 73, 59-66),structural changes in the basement membrane begin to occur in the latetwenties and have been found to accumulate with age. These structurechanges of basement membrane are skin changes that precede skin changesaccompanying aging, such as the formation of wrinkles, hardening ordecreased resiliency.

Thus, it is essential for expression of normal skin function thatepidermal basal cells be securely bound to the basement membrane, andpromotion of basement membrane formation is considered to be importantfor this. As a result of studying wrinkle formation inhibitors using anultraviolet radiation aged mouse model based on the presumption ofroutine ultraviolet rays, activity that inhibits wrinkle formation wasfound in drugs that are able to inhibit matrix metalloproteinase. Thus,agents that promote regeneration and repair of the basement membrane inthe skin are effective as inhibitors of skin aging.

Thus, according to the present invention, a skin activating compositionor basement membrane formation promoting artificial skin culturingliquid is newly provided that contains one type or a mixture of two ormore types of the above matrix metalloproteinase inhibiting compound, orone type or a mixture of two or more types of this matrixmetalloproteinase inhibiting compound and one type or a mixture of twoor more types of a compound that demonstrates activity which increasesthe production of matrix protein, at a concentration sufficient forexhibiting effects that promote regeneration, repair and formation ofskin basement membrane. This skin activation refers to, for example, theprevention or improvement of decreased skin function accompanyingstructural changes in basement membrane caused by aging and so forth,and more specifically, skin wrinkling and hardening.

The above concentration sufficient for exhibiting effects that promoteregeneration, repair and formation of skin basement membrane refers mayvary according to the type of compounds used, other components orvehicles used to prepare said composition, and the duration of use.

The skin basement membrane formation promoting agent of the presentinvention can contain active ingredient in the form of matrixmetalloproteinase inhibitor, or matrix metalloproteinase inhibitor andmatrix protein production promoting agent, at 0.000001 to 60 wt %, andpreferably 0.00001 to 60 wt %, relative to the composition.

The skin basement membrane stabilizing agent of the present inventioncan be in the form of an aqueous solution, oil, other solution, milkyliquid, cream, gel, suspension, microcapsules, powder, granules,capsules or solid, and after being prepared in any of these formsaccording to methods which are themselves known, can be coated, adhered,sprayed, injected, consumed or inserted into the body in the form of alotion, milky liquids, cream, ointment, paste, poultice, aerosol,injection, medication (including tablets, powders, granules, pills,syrups and troaches) or suppository. Among these preparation forms, skintopical preparations such as lotions, milky liquids, creams, ointments,pastes, poultices and aerosols are considered to be preparation formsthat are suitable for the composition of the present invention. The skintopical preparations described here include prescription drugs,non-prescription drugs and cosmetics, and are to be used with the samemeaning hereinafter.

Vehicles and fragrances routinely used when preparing such compositions,as well as oils, surfactants, antiseptics, sequestering agents,water-soluble polymers, thickeners, powder ingredients, ultravioletdefensive agents, moisturizing agents, pharmacologically effectiveingredients, antioxidants, pH adjusters, cleaning agents, drying agents,emulsifiers and so forth can be suitably blended into the composition ofthe present invention. In the case of blending each of these ingredientsinto the skin activating composition of the present invention, it isnecessary to blend them within a range that does not impair the desiredeffect of the present invention.

Examples of the above oil include liquid oils, solid oils, waxes,hydrocarbon oils, higher fatty acids, higher alcohols, synthetic esteroils and silicones.

More specifically, examples of liquid oils include avocado oil, tsubakioil, primrose oil, turtle oil, macadamia nut oil, corn oil, mink oil,olive oil, rape seed oil, egg yolk oil, sesame oil, persic oil, wheatgerm oil, sasanqua oil, castor oil, linseed oil, safflower oil,cottonseed oil, perilla oil, soy bean oil, peanut oil, theine oil, kayaoil, rice bran oil, Chinese wood oil, Japanese wood oil, hohoba oil,germ oil, triglycerin, trioctanoic glycerin and triisopalmitic glycerin;examples of solid oils include cacao butter, coconut oil, horse tallow,hardened coconut oil, palm oil, beef tallow, goat tallow, hardened beeftallow, palm heart oil, pork tallow, beef bone tallow, haze heart oil,hardened oil, beef leg tallow, haze wax and hardened castor oil;examples of waxes include beeswax, candelilla wax, cotton wax, carnaubawax, bayberry wax, tree wax, spermaceti, montan wax, bran wax, lanolin,kapok wax, lanolin acetate, liquid lanolin, sugar cane wax, isopropyllanolin fatty acid, hexyl laurate, reduced lanolin, jojoba wax, hardlanolin, shellac wax, POE lanolin alcohol ether, POE lanolin alcoholacetate, POE cholesterol ether, lanolin fatty acid polyethylene glycoland POE hydrogenated lanolin alcohol ether; and examples of hydrocarbonoils include liquid paraffin, ozokerite, squalene, pristan, paraffin,ceresin, squalene, Vaseline™ (petroleum jelly) and microcrystalline wax.

Examples of higher fatty acids include lauric acid, myristic acid,palmitic acid, stearic acid, behenic (beheninic) acid, oleic acid,12-hydroxystearic acid, undecylenic acid, tolic acid, isostearic acid,linoleic acid, linolenic acid, eicosapentanoic acid (EPA) anddocosahexanoic acid (DHA).

Examples of higher alcohols include lauryl alcohol, cetyl alcohol,stearyl alcohol, behenyl alcohol, myristyl alcohol, oleyl alcohol,cetostearyl alcohol and other straight chain alcohols, as well asmonostearyl glycerin ether (vatyl alcohol), 2-decyltetradecinol, lanolinalcohol, cholesterol, phytosterol, hexyldodecanol, isostearyl alcohol,octyldecanol and other branched chain alcohols.

Examples of synthetic ester oils include isopropyl myristate, cetyloctanoate, octyldodecyl myristate, isopropyl palmitate, butyl stearate,hexyl laurate, myristyl myristate, decyl oleate, hexyldecyldimethyloctanoate, cetyl lactate, myristyl lactate, lanolin acetate,isocetyl stearate, isocetyl isostearate, cholesteryl 12-hydroxystearate,ethylene glycol di-2-ethylhexylate, dipentaerythritol fatty acid ester,n-alkylglyol monoisostearate, glycerin di-2-heptylundecanoate,trimethylolpropane tri-2-ethylhexylate, trimethylolpropanetriisostearate, pentaneerythritol tetra-2-ethylhexylate, glycerintri-2-ethylhexylate, trimethylolpropane triisostearate, cetyl-2-ethylhexanoate, 2-ethylhexyl palmitate, glycerin trimyristate,tri-2-heptylundecanoic glyceride, castor oil fatty acid methyl ester,oleic oil, cetostearyl alcohol, acetoglyceride, 2-heptylundecylpalmitate, diisobutyl adipate, N-lauroyl-L-glutamate-2-octyldodecylester, di-2-heptylundecyl adipate, ethyl laurate, di-2-ethylhexylsebacate, 2-hexyldecyl myristate, 2-hexyldecyl palmitate, 2-hexyldecyladipate, diisopropyl sebacate, 2-ethylhexyl succinate, ethyl acetate,butyl acetate, amyl acetate and triethyl citrate.

Examples of silicones include dimethyl polysiloxane, methylphenylpolysiloxane, methylhydrogen polysiloxane and other linearpolysiloxanes, decamethyl polysiloxane, dodecamethyl polysiloxane,tetramethyltetrahydrogen polysiloxane and other cyclic polysiloxanes,and silicon resin and silicone rubber that form a three-dimensional meshstructure.

The skin basement membrane stabilizing agent of the present inventionmay contain anionic surfactant, cationic surfactant, amphotericsurfactant or nonionic surfactant and so forth either alone or incombination.

Examples of anionic surfactants include fatty acid soaps such as soapbase, sodium laurate and sodium palmitate; higher alkyl sulfate esterssuch as sodium lauryl sulfate and potassium lauryl sulfate; alkyl ethersulfate ester salts such as POE triethanol amine lauryl sulfate and POEsodium lauryl sulfate; N-acylsarcosinic acids such as sodiumlauroylsarcosine; higher fatty acid amide sulfonates such as sodiumN-myristoyl-N-taurine, sodium coconut oil fatty acid methyltauride andsodium laurylmethyltauride; phosphate esters such as POE sodium oleylether phosphate and POE stearyl ether phosphate; sulfosuccinates such assodium di-2-ethylhexylsulfosuccinate, sodium monolauroylmonoethanolamidepolyoxyethylene sulfosuccinate and sodium laurylpolypropylene glycolsulfosuccinate; alkylbenzene sulfonates such as linear sodiumdodecylbenzene sulfonate, linear dodecylbenzene sulfonatetriethanolamine and linear dodecylbenzene sulfonate; N-acylglutamatessuch as monosodium N-lauroylglutamate, disodium N-stearoylglutamate andmonosodium N-myristoyl-L-glutamate; higher fatty acid ester sulfateesters such as sodium hardened coconut oil fatty acid glycerin sulfate;sulfated oils such as Turkey red oil oil; as well as POE alkyl ethercarboxylic acid, POE alkylaryl ether carboxylic acid, α-olefinsulfonate, higher fatty acid ester sulfonate, secondary alcohol sulfateester, higher fatty acid alkylolamide sulfate ester, sodiumlauroylmonoethanolamide succinate, N-palmitoylaspartateditriethanolamine and sodium casein.

Examples of cationic surfactants include alkyl trimethyl ammonium saltssuch as stearyl trimethyl ammonium chloride and lauryl trimethylammonium chloride, dialkyl dimethyl ammonium salts such as distearyldimethyl ammonium chloride, alkyl pyridinium salts such as(N,N′-dimethyl-3,5-methylene piperidinium) chloride and cetyl pyridiniumchloride, alkyl quaternary ammonium salts, alkyl dimethylbenzyl ammoniumsalts, alkyl isoquinolinium salts, dialkyl morpholinium salts, POE alkylamines, alkyl amine salts, polyamine fatty acid derivatives, amylalcohol fatty acid derivatives, benzalkonium chloride and benzetoniumchloride.

Examples of amphoteric surfactants include imidazoline-based amphotericsurfactants such as2-undecyl-N,N,N-(hydroxyethylcarboxymethyl)-2-imidazoline sodium and2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy-2-sodium salt, andbetaine-based amphoteric surfactants such as2-heptadecyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine,lauryldimethylaminoacetate betaine, alkyl betaine, amidobetaine andsulfobetaine.

Examples of lipophilic nonionic surfactants include sorbitan fatty acidesters such as sorbitan monooleate, sorbitan monoisostearate, sorbitanmonolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitansesquioleate, sorbitan trioleate, penta-2-ethylhexylic diglycerolsorbitan and tetra-2-ethylhexylic diglycerol sorbitan, glycerinpolyglycerin fatty acids such as glycerin mono-cottonseed oil fattyacid, glycerin monoerucate, glycerin sesquiolate, glycerin monostearate,glycerin α,α′-oleic pyroglutamate, glycerin monostearate and malic acid,propylene glycol fatty acid esters such as propylene glycolmonostearate, hardened castor oil derivatives, glycerin alkyl ether andpolyoxyethylene-methylpolysiloxane copolymers.

Examples of hydrophilic nonionic surfactants include POE sorbitan fattyacid esters such as POE sorbitan monooleate, POE sorbitan monostearate,POE sorbitan monooleate and POE sorbitan tetraoleate, POE sorbitol fattyacid esters such as POE sorbitol monolaurate, POE sorbitol monooleate,POE sorbitol pentaoleate and POE sorbitol monostearate, POE glycerinfatty acid esters such as POE glycerin monostearate, POE glycerinmonoisostearate and POE glycerin triisostearate, POE fatty acid esterssuch as POE monooleate, POE distearate, POE monodioleate and ethyleneglycol distearate, POE alkyl ethers such as POE lauryl ether, POE oleylether, POL stearyl ether, POE behenyl ether, POE 2-octyldodecyl etherand POE cholestanol ether, POE alkylphenyl ethers such as POEoctylphenyl ether, POE nonylphenyl ether and POE dinonylphenyl ether,plurallonics such as pluronic, POE-POP alkyl ethers such as POE-POPcetyl ether, POE-POP 2-decyltetradecyl ether, POE-POP monobutyl ether,POE-POP hydrogenated lanolin and POE-POP glycerin ether, tetra POE-tetraPOP ethylene diamine condensation products such as tetronic, POE castoroil/hardened castor oil derivatives such as POE castor oil, POE hardenedcastor oil, POE hardened castor oil monoisostearate, POE hardened castoroil triisostearate, POE hardened castor oil monopyroglutamatemonoisostearate diester and POE hardened castor oil maleate, POEbeeswax-lanolin derivatives such as POE sorbitol beeswax, alkanol amidessuch as coconut oil fatty acid diethanol amide, lauric monoethanol amideand fatty acid isopropanol amide, POE propylene glycol fatty acidesters, POE alkyl amines, POE fatty acid amides, sucrose fatty acidesters, POE nonylphenyl formaldehyde compounds, alkylethoxydimethylamine oxides and trioleyl phosphates.

Examples of the above preservatives include methyl parabenzene, ethylparabenzene and butyl parabenzene.

Examples of the above sequestering agents include sodium edetate andEDTA.

Examples of the above water-soluble polymers include natural polymers,semi-synthetic polymers, synthetic polymers and inorganic polymers.

Examples of natural water-soluble polymers include plant polymers suchas gum arabic, tragacanth gum, galactan, guar gum, carob gum, karayagum, carageenan, tamarind gum, xanthane gum, pectin, agar, queenseedmarmelo, algea colloid (brown algae extract), starch (rice, corn,potato, wheat) and glycyrrhizic acid, microbial polymers such asxanthane gum, dextran succinoglucan and pluran, and animal polymers suchas collagen, casein, albumin and gelatin.

Examples of semi-synthetic water-soluble polymers include starch-basedpolymers such as dextrin, carboxymethyl starch and methylhydroxypropylstarch, cellulose-based polymers such as methyl cellulose,nitrocellulose, ethyl cellulose, methylhydroxypropyl cellulose,hydroxyethyl cellulose, cellulose dimethyl dialkyl (12-20) ammoniumsulfate, hydroxypropyl cellulose, sodium carboxymethyl cellulose (CMC),crystalline cellulose and powdered cellulose, alginate-based polymerssuch as sodium alginate and propylene glycol alginate ester.

Examples of synthetic water-soluble polymers include vinyl-basedpolymers such as polyvinyl alcohol, polyvinyl methyl ether, polyvinylpyrrolidone, carboxyvinyl polymer and alkyl denatured carboxyvinylpolymer, polyoxyethylene-based polymers such as polyethylene glycol2000, 4000 and 6000, polyoxyethylene polyoxypropylene copolymer-basedpolymers, acrylic polymers such as sodium polyacrylate, polyethyleneacrylate and polyacrylamide, polyethylene imines and cationic polymers.

Examples of inorganic water-soluble polymers include bentonite,magnesium aluminum silicate, raponite, hectrite and silicic anhydride.

Examples of the above powder component include inorganic powders such astalc, kaolin, mica, sericite, muscovite, phlogopite, synthetic mica,lepidolite, biotite, lithia mica, vermiculite, magnesium carbonate,calcium carbonate, aluminum silicate, barium silicate, calcium silicate,magnesium silicate, strontium silicate, metal tungstate, magnesium,silica, zeolite, barium sulfate, baked calcium sulfate (baked gypsum),calcium phosphate, fluoroapatite, hydroxyapatite, ceramic powder, metalsoap (zinc myristate, calcium palmitate, aluminum stearate) and boronnitride, organic powders such as polyamide resin powder (Nylon powder),polyethylene powder, polymethyl methacrylate powder, polystyrene powder,styrene and acrylic acid copolymer resin powder, benzoguanamine resinpowder, polytetrafluoroethylene powder and cellulose powder, inorganicwhite pigments such as titanium dioxide and zinc oxide, inorganic redpigments such as iron oxide (rouge) and iron titanate, inorganic brownpigments such as γ-iron oxide, inorganic yellow pigments such as yellowiron oxide and ocher, inorganic black pigments such as black iron oxide,carbon black and low order titanium oxide, inorganic violet pigmentssuch as mango violet and cobalt violet, inorganic green pigments such aschromium oxide, chromium hydroxide and cobalt titanate, inorganic bluepigments such as ultramarine and Prussian blue, pearl pigments such astitanium oxide coated mica, titanium oxide coated bismuth oxychloride,titanium oxide coated talc, colored titanium oxide coated mica, bismuthoxychloride and fish scale foil, metal powder pigments such as aluminumpowder and copper powder, zirconium, barium or aluminum lake organicpigments such as red no. 201, red no. 202, red no. 204, red no. 205, redno. 220, red no. 226, red no. 228, red no. 405, orange no. 203, orangeno. 204, yellow no. 205, yellow no. 401, yellow no. 404, red no. 3, redno. 104, red no. 106, red no. 227, red no. 230, red no. 401, red no.505, orange no. 205, yellow no. 4, yellow no. 5, yellow no. 202, yellowno. 203, green no. 3 and blue no. 1, natural pigments such aschlorophyll and β-carotene, and colorants such as titanium yellow,carsamine and saffron red.

Examples of the above ultraviolet defensive agents include both“ultraviolet absorbers”, which are substances that chemically absorbultraviolet rays, and “ultraviolet blockers”, which are substances thatscatter and reflect ultraviolet rays by physical action.

Namely, examples of long-wavelength ultraviolet (UVA) absorbers includeanthranilic acid-based ultraviolet absorbers such as methyl anthranilateand homomenthyl-N-acetylanthranilate, benzophenone-based ultravioletabsorbers such as 2,4-dihydroxybenzophenone,2,2-dihydroxy-4-methoxybenzophenone,2,2′-dihydroxy-4,4′-dimethoxybenzophenone,2,2′,4,4′-tetrahydroxybenzophenone, 2-hydroxy-4-methoxybenzophenone,2-hydroxy-4-methoxy-4′-methylbenzophenone,2-hydroxy-4-methoxybenzophenone-5-sulfonic acid, 4-phenylbenzophenone,2-ethylhexyl-4′-phenyl-benzophenone-2-carboxylate,2-hydroxy-4-n-octoxybenzophenone and 4-hydroxy-3-carboxybenzophenone,benzotriazole-based ultraviolet absorbers such as2,2′-hydroxy-5-methylphenylbenzotriazole,2-(2′-hydroxy-5′-t-octylphenyl)benzotriazole and2-(2′-hydroxy-5′-methylphenyl)benzotriazole, dianisoylmethane and4-methoxy-4′-t-butyldibenzoylmethane.

Among these long-wavelength ultraviolet absorbers,4-methoxy-4′-tert-butyldibenzoylmethane, 2-hydroxy-4-methoxybenzophenoneand 2-hydroxy-4-methoxybenzophenone derivatives such as2-hydroxy-4-methoxybenzophenone-5-sulfonate are preferable because theyare long-wavelength ultraviolet absorbers having superior safety andeffectiveness.

In addition, examples of medium-wavelength ultraviolet (UVB) absorbersinclude benzoic acid-based ultraviolet absorbers such as paraminobenzoicacid (to be abbreviated as PABA), PABA monoglycerin ester, N,N-dipropoxyPABA ethyl ester, N,N-diethoxy PABA ethyl ester, N,N-dimethyl PABA ethylester, N,N-dimethyl PABA butyl ester and N,N-dimethyl PABA amyl ester,salicylic acid-based ultraviolet absorbers such as dipropylene glycolsalicylate, ethylene glycol salicylate, myristyl salicylate, methylsalicylate, amyl salicylate, menthyl salicylate, homomenthyl salicylate,octyl salicylate, phenyl salicylate, benzyl salicylate and p-isopropanolphenyl salicylate, cinnamic acid-based ultraviolet absorbers such asoctyl cinnamate, ethyl-4-isopropyl cinnamate, methyl-2,5-diisopropylcinnamate, ethyl-2,4-diisopropyl cinnamate, methyl-2,4-diisopropylcinnamate, propyl-p-methoxycinnamate, isopropyl-p-methoxycinnamate,isoamyl-p-methoxycinnamate, octyl-p-methoxycinnamate(2-ethylhexyl-p-methoxycinnamate), 2-ethoxymethyl-p-methoxycinnamate,cyclohexyl-p-methoxycinnamate, ethyl-α-cyano-β-phenylcinnamate,2-ethylhexyl-α-cyano-β-phenylcinnamate,glycerylmono-2-ethylhexanoyl-diparamethoxycinnamate, methoxycinnamateoctyl,3,4,5-trimethoxycinnamate-3-methyl-4-[methylbis(trimethylsiloxy)silyl]butyland p-dimethoxycinnamate monoethyl ester, camphor derivatives such as3-(4′-methylbenzylidene)-d,1-camphor, 3-benzylidene-d,1-camphor and5-(3,3-dimethyl-2-norvolnilidene)-3-penten-2-one, urocanic acid,urocanic ethyl ester, 2-phenyl-5-methylbenzooxazole and dibenzaladine.

Moreover, examples of ultraviolet blockers include titanium oxide(TiO₂), talc (MgSiO₂), carmine (FeO₂), bentonite, kaolin and zinc oxide(ZnO).

Examples of the above moisturizing agents include polyethylene glycol,propylene glycol, glycerin, 1,3-butylene glycol, hexylene glycol,xylitol, sorbitol, maltitol, chondroitin sulfuric acid, hyaluronic acid,mucoitin sulfuric acid, charoninic acid, atherocollagen,cholesteryl-12-hydroxystearate, sodium lactate, bile acid salt,d1-pyrrolidone carboxylate, short chain soluble collagen, diglycerin(EO) PO addition product, Chestnut rose Fruit extract, milfoil extractand melilote extract.

Examples of the above pharmacologically effective ingredients includewhiteners such as albutin, vitamin C and its derivatives, kojic acidplacental extract, glutathione and saxifrage extract, antiphlogisticssuch as glycyrrhizic acid derivatives, glycyrrhetinic acid derivatives,salicylic acid derivatives, hinokitiol, zinc oxide and allantoin,activators such as royal jelly, actinolite, cholesterol derivatives andcalf blood extract, circulation promoters such as nonylic urenylamide,nicotinic benzyl ester, nicotinic β-butoxyethyl ester, capsisin,zingerone, cantharidin, ichthammol, caffeine, tannic acid, α-borneol,nicotinic tocopherol, inositol hexanicotinate, cyclandelate,cinnarizine, tolazoline, acetylcholine, verapamil, cepharanthin andγ-olizanol, antiseborrheics such as sulfur and thianthol, and forvarious purposes, Phellodendron Bark extract component, goldthreadextract component, Lithospermum Root extract component, peony extractcomponent, gentian extract component, birch extract component, sageextract component, loquat extract component, carrot extract component,aloe extract component, mallow extract component, iris extractcomponent, grape extract component, Coix Seed extract component, spongecucumber extract component, lily extract component, saffron extractcomponent, Cnidium Officinale Root extract component, Ginger Rootextract component, Saint John's-wort extract component, Restharrow Rootextract component, rosemary extract component, garlic extract component,red pepper extract component, dried orange peel, Japanese angelica rootand so forth, vitamin A substances such as retinol and retinol acetate,vitamin B₂ substances such as riboflavin butyrate and riboflavin adeninenucleotide, vitamin B2 substances such as pyridoxine hydrochloride andpyridoxine dioctanoate, vitamin C substances such as L-ascorbic acid,L-ascorbate dipalmitate ester, L-ascorbate-2-sodium sulfate, L-ascorbatephosphate ester and dipotassium DL-α-tocopherol-L-ascorbate phosphatediester, pantothenic acids such as calcium pantothenate, D-pantothenylalcohol, pantothenyl ethyl ether and acetylpentothenyl ethyl ether,vitamin D substances such as ergocalciferol and cholecalciferol,nicotinic acids such as nicotinic acid, nicotinic amide and benzylnicotinate, vitamin E substances such as α-tocopherol andDL-α-tocopherol succinate, and other vitamins such as vitamin P andbiotin.

Furthermore, these pharmacological components can be widely blendedwithin a range over which the desired pharmacological effect of thepresent invention is not impaired by that blending.

The composition of the present invention prepared in this mannerprevents decreased function accompanying structural changes of basementmembrane, and promotes activation of the skin.

EXAMPLES

The following provides a more detailed explanation of the presentinvention through its examples.

Example 1 Production of Artificial Skin Using Matrix MetalloproteinaseInhibitor

Collagen gel was obtained by preparing 10 ml of a collagen solution ofsuspended fibroblasts (0.3 to 1×10⁵ cells/ml) originating in humandermis (product I-AC of Koken Co., Ltd. was used for the collagen) onice followed by gelling the collagen at 37° C. in a 60 mm Petri dish.Next, the gel was peeled from the walls of the Petri dish and thecollagen gel was contracted in a CO₂ incubator.

This contracted collagen gel equivalent to dermis was placed on a metalmesh and a glass ring (inner diameter: 12 mm) was placed over this gel.0.4 ml of a suspension of epidermal keratinocytes (1×10⁶/ml) (mixedmedium of KGM-DMEM containing 5% fetal calf serum (1:1)) were placedinside the glass ring to prevent leakage of liquid. After adheringkeratinocytes in a CO₂ incubator overnight, the ring was removed on thefollowing day. The above medium was filled to the boundary of theepidermal layer and culturing was continued while exposing the epidermallayer to air to produce a skin model having a stratified epidermisexhibiting corneal layer formation.

After inoculating the epidermic cells, the medium was changed to thatcontaining either (1) 10 μM CGS27023A (matrix metalloproteinaseinhibitor) or (2) 300 μM MMP-inhibitor(p-NH₂-Bz-Gly-Pro-D-Leu-D-Ala-NHOH (FN-437) (BBRC, 1994, 199, p.1442-1446) (acquired from Cabbiochem. Novabiochem Corporation) (matrixmetalloproteinase inhibitor)) starting in the first week, and thenreplaced with medium containing the same type and same concentration ofmatrix metalloproteinase inhibitor every 2-3 days after that andcultured for 2 weeks. In addition, (3) similar culturing was carried outwithout adding matrix metalloproteinase inhibitor for use as a control.

Those results are shown in FIG. 1. As is clear from the figure, althougha basement membrane-like structure was not observed directly beneath thebasal cells of the epidermis in control (3), in the case of addingmatrix metalloproteinase inhibitors (1) CGS27023A compound or (2)MMP-inhibitor, promotion of basement membrane formation was clearlyobserved.

A similar experiment was carried out for extracts of the plantsindicated below instead of the above matrix metalloproteinase inhibitorcompounds to confirm promotion of basement membrane formation.

Similar effects were confirmed for Thymus serpyllum L., Valerianafauriei Briquet or other similar plants (Valerianaceae), Diospyros kakiThunberg (Ebenaceae), Astragalus sinicus Linne (Leguminosae), Crataeguscuneata Siebold et Zuccarini (Rosaceae), Paeonia suffruticosa Andrews(Poeonia montan Sims) (Paconiaceae), Thea sinensis Linne var. assamicaPierre (Thcaccae), Eucalyptus globulus Labillardiere or its similarplants (Myrtaceae), Potentilla tormentilla Schrk (Rosaceae), Tiliacorda: a Mill., Tilia platyphyllus Scop., Tilia europaea Linne(Tiliaceae), Betula alba Linne (Betulaceze), Origanum majorana L.,Uncaria gambir Roxburgh (Rubiaceae), Juglans regia Linne var. sinensisDe Candolie or its similar plants (Juglandaceae), Sophora flavescensAiton (Leguminosae), Sanguisorba officinalis Linne (Rosaceae), Hypericumperforatum Linne or Hypericum erectum Thunberg (Guttiferae), Theasinensis Linne (Theaceae), Curcuma longa L (Zingiberaceae), purifiedextracts of Curcuma longa L including Symplocos racemosa and Cyperusrotundus, Cyperpus scariosus, Gaultheria fragrantissima, Acaciaformensia, Terminalia chebula, Ficus bengalensis, Cassia fistula Linn,Lyonia ovalifolia, Calophyllum inophyllum and Ficus religiosa.

Example 2 Production of Artificial Skin Using Both MatrixMetalloproteinase Inhibitor and Matrix Protein Production Promoting

Agent

Although the same method as Example 1 was repeated, the followingsubstances were used as test substances.

-   (1) 10 μM matrix metalloproteinase inhibitor CGS27023A only;-   (2) 10 μM matrix metalloproteinase inhibitor CGS27023A and 10 ng/ml    of matrix metalloproteinase protein production promoting agent,    Transforming Growth Factor α (TGFa);-   (3) 10 μM matrix metalloproteinase inhibitor CGS27023A and 10 ng/ml    of matrix protein production promoting agent, Transforming Growth    Factor β1 (TGFb1);-   (4) 10 μM matrix metalloproteinase inhibitor CGS27023A, 10 ng/ml of    matrix protein production promoting agent, Transforming Growth    Factor α (TGFa) and 10 ng/ml of Transforming Growth Factor β1    (TGFb1); and-   (5) culturing of a control to which none of the above were added.

The formed artificial skin was stained by hematoxylin-eosin (H & E) andimmunostaining (using anti-laminin 5 (LN5) antibody, anti-type IVcollagen (IVC) antibody, and anti-type VII collagen (VIIC) antibody).Those results are shown in FIG. 2, FIG. 3 and Table 1. Furthermore, theresults for H & E and LN5 are shown in FIG. 2, while the results for IVCand VIIC are shown in FIG. 3.

In the control, adhesion of epidermis/dermis joined portions was weak,the epidermis easily peeled from the dermis and there were voidsobserved in the tissue. In contrast, in the group treated with matrixmetalloproteinase inhibitor CGS27023A, adhesion between the epidermisand dermis was clear. In addition, in the case of adding matrix proteinproduction promoting agent, Transforming Growth Factor α (TGFa),Transforming Growth Factor β1 (TGFb1) or both (TGFa+TGFb1) in additionto matrix metalloproteinase inhibitor CGS27023A, staining of type VIIcollagen in particular was promoted significantly, thereby confirmingthat adhesion between the epidermis and dermis was further promoted.TABLE 1 Effects of Various Drugs on Basement Membrane Formation in aSkin Model Deposition of basement membrane component Basement TreatmentType IV Type VII membrane conditions Laminin 5 collagen collagenformation Non-treatment + + − − control group CGS27023A ++ ++ + +CGS27023A + ++ ++ ++ ++ soybean lecithin CGS27023A + TGFα ++ ++ ++CGS27023A + TGFβ1 ++ ++ ++ CGS27023A + ++ ++ ++ TGFα + TGFβ1

Culturing was carried out in the same manner as above using thefollowing plant extracts instead of the above matrix metalloproteinaseinhibitor CGS27023A.

Thymus serpyllum L., Valeriana fauriei Briquet or other similar plants(Valerianaceae), Diospyros kaki Thunberg (Ebenaceae), Astragalus sinicusLinne (Leguminosae), Crataegus cuneata Siebold et Zuccarini (Rosaceae),Paeonia suffruticosa Andrews (Poeonia montan Sims) (Paconiaceae), Theasinensis Linne var. assamica Pierre (Thcaccae), Eucalyptus globulusLabillardiere or its similar plants (Myrtaceae), Potentilla tormentillaSchrk (Rosaceae), Tilia corda: a Mill., Tilia platyphyllus Scop., Tiliaeuropaea Linne (Tiliaceae), Betula alba Linne (Betulaceze), Origanummajorana L., Uncaria gambir Roxburgh (Rubiaceae), Juglans regia Linnevar. sinensis De Candolie or its similar plants (Juglandaceae), Sophoraflavescens Aiton (Leguminosae), Sanguisorba officinalis Linne(Rosaceae), Hypericum perforatum Linne or Hypericum erectum Thunberg(Guttiferae), Thea sinensis Linne (Theaceae), Curcuma longa L(Zingiberaceae), purified extracts of Curcuma longa L includingSymplocos racemosa and Cyperus rotundus, Cyperus scariosus, Gaultheriafragrantissima, Acacia formensia, Terminalia chebula, Ficus bengalensis,Cassia fistula Linn, Lyonia ovalifolia, Calophyllum inophyllum and Ficusreligiosa.

As a result, results were obtained that were similar to the case ofusing CGS27023A as matrix metalloproteinase inhibitor. In addition,similar results were also obtained for Symplocos racemosa, a purifiedextract of Curcuma longa L.

Moreover, a structure resembling a basement membrane was continuouslyobserved directly beneath the basal cells in the case of using 30 μg/mlof soy bean lecithin instead of Transforming Growth Factor for thematrix protein production promoting agent. Those results are shown inFIG. 4.

Example 3 Preparation of Artificial Skin Culture Medium

Matrix metalloproteinase inhibitor CGS27023A or MMP-inhibitor were addedto a concentration of 10 μM to basal medium comprising a 1:1 mixture ofKGM (keratinocyte growth medium), typically used to culture epidermiccells, and Dulbecco's Modified Eagle's Medium containing 10% fetal calfserum, typically used to culture fibroblasts. Furthermore, KGM wasprepared by adding HEPES (6.7 g/L), NaHCO₃ (1.2 g/L), insulin (5 mg/L),transferrin (10 mg/L), hydrocortisone (0.5 mg/L) andphosphorylethanolamine (14.1 mg/L) to MCDB153 medium (10.93 g/L) andadjusting the pH to 7.4 followed by the addition of 20 g of bovinepituitary extract (Far East Pharmaceutical).

Example 4 Preparation of Artificial Skin Culture Medium

At least one type of Transforming Growth Factor α (TGFa) or TransformingGrowth Factor β1 (TGFb1) were added as matrix protein productionpromoting agent to the artificial skin culture medium in Example 3 to aconcentration of 10 ng/ml.

In addition, soy bean lecithin was added at 30 μg/ml in place of theabove TGFa or TGFb1.

Preparation Example 1 Cream

Polyoxyethylene (addition of 20 moles) cetyl alcohol ether 1.0Methylphenyl polysiloxane (20 cs) 2.0 Liquid paraffin 3.02-hydroxy-4-methoxybenzophenone 5.0 Active ingredient 0.2 Propyleneglycol 5.0 Glycerin 2.0 Ethyl alcohol 15.0 Carboxyvinyl polymer 0.3Hydroxypropyl cellulose 0.1 2-aminomethylpropanol 0.1 Antiseptic Assuitable Fragrance As suitable Ion exchanged water As suitable

(Production Method)

Propylene glycol, glycerin, ethyl alcohol, carboxyvinyl polymer,hydroxypropyl cellulose and 2-aminomethylpropanol were added to ionexchanged water followed by heating to 70° C. (aqueous phase).

Methylphenyl polysiloxane, liquid paraffin, polyoxyethylene cetylalcohol ether, antiseptic, 2-hydroxy-4-methoxybenzophenone, activeingredient (such as lipidure and/or CGS27023A) and fragrance were mixedand prepared to 70° C. (oily phase).

After gradually adding the aqueous phase to the oily phase,pre-emulsifying and obtaining homogeneous emulsified particles using ahomomixer, the emulsion was deaerated and cooled to obtain a cream.

Preparation Example 2 Cream

Polyoxyethylene (addition of 20 moles) cetyl alcohol ether 1.0Methylphenyl polysiloxane (20 cs) 2.0 Liquid paraffin 3.02-hydroxy-4-methoxybenzophenone 5.0 Active ingredient 0.2 Propyleneglycol 5.0 Glycerin 2.0 Ethyl alcohol 15.0 Carboxyvinyl polymer 0.3Hydroxypropyl cellulose 0.1 2-aminomethylpropanol 0.1 Antiseptic Assuitable Fragrance As suitable Ion exchanged water As suitable

(Production Method)

Propylene glycol, glycerin, ethyl alcohol, carboxyvinyl polymer,hydroxypropyl cellulose and 2-aminomethylpropanol were added to ionexchanged water followed by heating to 70° C. (aqueous phase).

Methylphenyl polysiloxane, liquid paraffin, polyoxyethylene cetylalcohol ether, antiseptic, 2-hydroxy-4-methoxybenzophenone, activeingredient (such as lipidure and/or CGS27023A) and fragrance were mixedand prepared to 70° C. (oily phase).

Preparation Example 3 Milky Liquid

Wt % Cetyl alcohol 1.0 Beeswax 0.5 Vaseline (Petroleum Jelly) 2.0Squalene 6.0 Dimethyl polysiloxane 2.0 Ethyl alcohol 5.0 Glycerin 4.01,3-butylene glycol 4.0 Active ingredient 0.1 Tranexamic acid 1.0Polyoxyethylene (10) monooleic ester 1.0 Glycerol monostearic ester 1.0Queenseed extract (5% aqueous solution) 20.0 Antiseptic As suitableFragrance As suitable Ion exchanged water As suitable

(Production Method)

Glycerin and 1,3-butylene glycol were added to ion exchanged water,mixed and heated to 70° C. (aqueous phase). Cetyl alcohol, beeswax,Vaseline (petroleum jelly), squalene, dimethyl polysiloxane, activeingredient (such as lipidure or CGS27023A), tranexamic acid,polyoxyethylene (10) monooleic ester, glycerol monostearic ester andantiseptic were prepared in the form of a mixture and heated to 70° C.(oily phase). After adding the aqueous phase to the oily phase andperforming preliminary emulsification, adding and stirring queenseedextract and ethyl alcohol and obtaining homogeneous emulsified particlesusing a homomixer, the emulsion was deaerated, filtered and cooled toobtain a milky liquid.

Preparation Example 4 Milky Liquid

Wt % Cetyl alcohol 1.0 Beeswax 0.5 Vaseline (Petroleum Jelly) 2.0Squalene 6.0 Dimethyl polysiloxane 2.0 Ethyl alcohol 5.0 Glycerin 4.01,3-butylene glycol 4.0 Active ingredient 0.1 Tranexamic acid 1.0Polyoxyethylene (10) monooleic ester 1.0 Glycerol monostearic ester 1.0Queenseed extract (5% aqueous solution) 20.0 Antiseptic As suitableFragrance As suitable Ion exchanged water As suitable

(Production Method)

Glycerin and 1,3-butylene glycol were added to ion exchanged water,mixed and heated to 70° C. (aqueous phase). Cetyl alcohol, beeswax,Vaseline (petroleum jelly), squalene, dimethyl polysiloxane, activeingredient (such as lipidure or CGS27023A), tranexamic acid,polyoxyethylene (10) monooleic ester, glycerol monostearic ester andantiseptic were prepared in the form of a mixture and heated to 70° C.(oily phase). After adding the aqueous phase to the oily phase andperforming preliminary emulsification, adding and stirring queenseedextract and ethyl alcohol and obtaining homogeneous emulsified particlesusing a homomixer, the emulsion was deaerated, filtered and cooled toobtain a milky liquid.

After gradually adding the aqueous phase to the oily phase,pre-emulsifying and obtaining homogeneous emulsified particles using ahomomixer, the emulsion was deaerated and cooled to obtain a cream.

1. A method for promoting a skin basement membrane formation in asubject for which the skin basement membrane formation is necessary,comprising administering a matrix metalloproteinase inhibitorN-hydroxy-2(R)-[[(4-methoxyphenyl)sulfonyl](3-picolyl)amino]-3-methylbutanamidehydrochloride to said subject.
 2. A method for promoting a skin basementmembrane formation in a subject for which the skin basement membraneformation is necessary, comprising administering a matrixmetalloproteinase inhibitorN-hydroxy-2(R)-[[(4-methoxyphenyl)sulfonyl](3-picolyl)amino]-3-methylbutanamidehydrochloride and a matrix protein production promoting agent to saidsubject. 3-6. (canceled)
 7. The method for promoting a skin basementformation in accordance with claim 1, further comprising mixing saidmatrix metalloproteinase inhibitor with a matrix metalloproteinaseinhibitor selected from the group consisting of Thymus serpyllum L.,Valeriana fauriei Briquet or other similar plants (Valerianaceae),Diospyros kaki Thunberg (Ebenaceae), and Astragalus sinicus Linne(Leguminosae).
 8. The method for promoting a skin basement formation inaccordance with claim 1, further comprising mixing said matrixmetalloproteinase inhibitor with a matrix metalloproteinase inhibitorselected from the group consisting of Crataegus cuneata Siebold etZuccarini (Rosaceae), Paeonia suffruticosa Andrews (Poeonia montan Sims)(Paconiaceae), Thea sinensis Linne var. assamica Pierre (Thcaccae), andEucalyptus globules Labillardiere or its similar plants (Myrtaceae). 9.The method for promoting a skin basement formation in accordance withclaim 1, further comprising mixing said matrix metalloproteinaseinhibitor with a matrix metalloproteinase inhibitor selected from thegroup consisting of Potentilla tormentilla Schrk (Rosaceae), Tiliacorda: a Mill., Tilia platyphyllus Scop., and Tilia europaea Linne(Tiliaceae).
 10. The method for promoting a skin basement formation inaccordance with claim 1, further comprising mixing said matrixmetalloproteinase inhibitor with a matrix metalloproteinase inhibitorselected from the group consisting of Betula alba Linne (Betulaceze),Origanum majorana L., Uncaria gambir Roxburgh (Rubiaceae), and Juglansregia Linne var. sinensis De Candolie or its similar plants(Juglandaceae).
 11. The method for promoting a skin basement formationin accordance with claim 1, further comprising mixing said matrixmetalloproteinase inhibitor with a matrix metalloproteinase inhibitorselected from the group consisting of Sophora flavescens Aiton(Leguminosae), Sanguisorba officinalis Linne (Rosaceae), Hypericumperforatum Linne or Hypericum erectum Thunberg (Guttiferae), and Theasinensis Linne (Theaceae).
 12. The method for promoting a skin basementformation in accordance with claim 1, further comprising mixing saidmatrix metalloproteinase inhibitor with a matrix metalloproteinaseinhibitor selected from the group consisting of Curcuma longa L(Zingiberaceae), purified extracts of Curcuma longa L includingSymplocos racemosa and Cyperus rotundus, Cyperus scariosus, Gaultheriafragrantissima, and Acacia formensia.
 13. The method for promoting askin basement formation in accordance with claim 1, further comprisingmixing said matrix metalloproteinase inhibitor with a matrixmetalloproteinase inhibitor selected from the group consisting ofTerminalia chebula, Ficus bengalensis, Cassia fistula Linn, Lyoniaovalifolia, Calophyllum inophyllum, and Ficus religiosa.
 14. The methodfor promoting a skin basement formation in accordance with claim 2,further comprising mixing said matrix metalloproteinase inhibitor with amatrix metalloproteinase inhibitor selected from the group consisting ofThymus serpyllum L., Valeriana fauriei Briquet or other similar plants(Valerianaceae), Diospyros kaki Thunberg (Ebenaceae), and Astragalussinicus Linne (Leguminosae).
 15. The method for promoting a skinbasement formation in accordance with claim 2, further comprising mixingsaid matrix metalloproteinase inhibitor with a matrix metalloproteinaseinhibitor selected from the group consisting of Crataegus cuneataSiebold et Zuccarini (Rosaceae), Paeonia suffruticosa Andrews (Poeoniamontan Sims) (Paconiaceae), Thea sinensis Linne var. assamica Pierre(Thcaccae), and Eucalyptus globules Labillardiere or its similar plants(Myrtaceae).
 16. The method for promoting a skin basement formation inaccordance with claim 2, further comprising mixing said matrixmetalloproteinase inhibitor with a matrix metalloproteinase inhibitorselected from the group consisting of Potentilla tormentilla Schrk(Rosaceae), Tilia corda: a Mill., Tilia platyphyllus Scop., and Tiliaeuropaea Linne (Tiliaceae).
 17. The method for promoting a skin basementformation in accordance with claim 2, further comprising mixing saidmatrix metalloproteinase inhibitor with a matrix metalloproteinaseinhibitor selected from the group consisting of Betula alba Linne(Betulaceze), Origanum majorana L., Uncaria gambir Roxburgh (Rubiaceae),and Juglans regia Linne var. sinensis De Candolie or its similar plants(Juglandaceae).
 18. The method for promoting a skin basement formationin accordance with claim 2, further comprising mixing said matrixmetalloproteinase inhibitor with a matrix metalloproteinase inhibitorselected from the group consisting of Sophora flavescens Aiton(Leguminosae), Sanguisorba officinalis Linne (Rosaceae), Hypericumperforatum Linne or Hypericum erectum Thunberg (Guttiferae), and Theasinensis Linne (Theaceae).
 19. The method for promoting a skin basementformation in accordance with claim 2, further comprising mixing saidmatrix metalloproteinase inhibitor with a matrix metalloproteinaseinhibitor selected from the group consisting of Curcuma longa L(Zingiberaceae), purified extracts of Curcuma longa L includingSymplocos racemosa and Cyperus rotundus, Cyperus scariosus, Gaultheriafragrantissima, and Acacia formensia.
 20. The method for promoting askin basement formation in accordance with claim 2, further comprisingmixing said matrix metalloproteinase inhibitor with a matrixmetalloproteinase inhibitor selected from the group consisting ofTerminalia chebula, Ficus bengalensis, Cassia fistula Linn, Lyoniaovalifolia, Calophyllum inophyllum and Ficus religiosa.
 21. The methodfor promoting a skin basement formation in accordance with claim 1,further comprising mixing said matrix metalloproteinase inhibitor with amatrix metalloproteinase inhibitor selected from the group consisting ofSymplocos racemosa, a purified extract of Curcuma longa L.
 22. Themethod for promoting a skin basement formation in accordance with claim2, further comprising mixing said matrix metalloproteinase inhibitorwith a matrix metalloproteinase inhibitor selected from the groupconsisting of Symplocos racemosa, a purified extract of Curcuma longa L.23. The method for promoting skin basement formation in accordance withclaim 2, wherein said matrix protein production promoting agent isselected from the group consisting of one or more of soybeanlysolecithin transforming growth factor α (TGFa), and transforminggrowth factor β1 (TGFb1).